Avian infectious coryza is one of the most important respiratory diseases in poultry, which is an acute respiratory disease caused by infection with Haemophilus paragallinarum (hereinafter also referred to as “HPG”) with cardinal symptoms being a running nose, swelling of the face and epiphora. Avian infectious coryza brings about a great economical damage since it leads to decrease in the breeding rate of poultry, retarding of egg laying, decrease in egg production or failure of egg laying. For prevention of avian infectious coryza, an inactivated vaccine has hitherto used widely which is obtained by culturing Haemophilus paragallinarum, recovering and inactivating the cells with formalin, thimerosal and the like. However, adverse side effects caused by such an inactivated vaccine has been an issue as it has been reported that local necrotic lesions are formed in the inoculated chicken when the vaccine is administered (M. Matsumoto, et al. (1971)), and hence, development of a highly safe vaccine is earnestly desired.
In recent years, laborsaving in breeding and managing poultry is in progress with a scale-up of breeding poultry. As a part of this, laborsaving in vaccination has also been earnestly desired, and as a result, a mixed vaccine has already been developed and widely used in the field so that a frequency of inoculation can be reduced by mixing several kinds of vaccines together.
In order to provide a mixed vaccine showing immunogenicity equivalent to that of each plain vaccine without increase of dosage amount, it is necessary to increase an amount of each antigen contained in a mixed vaccine or to find out and use a more suitable adjuvant. However, in case of gram-negative bacteria such as HPG, a higher amount of antigen is likely to enhance a response to injection such as swelling at the inoculated site. Therefore, in order to reduce such an adverse response, it is preferable to obtain only a protective antigen, i.e. an effective component, from bacterial cells or culture supernatant, or to clone a gene coding for said antigen by the genetic recombination technique, to express said gene in bacteria, yeast, an animal cell, a plant cell, an insect cell and the like, and to purify a product expressed in a large amount, which is then mixed with an appropriate adjuvant together with other vaccines.
Another approach for laborsaving of vaccination is the use of virus or bacteria as a vector. That is, genes coding for protective antigens from one or plural pathogens have been incorporated into an attenuated virus or bacteria to prepare a polyvalent live vaccine. For fowls, poxvirus, Marek's disease virus and the like have been investigated as a vector. A vaccine comprising a viral vector has been put into practice wherein genes coding for HN and F proteins of Newcastle disease virus are incorporated into fowl pox virus.
It is thus most important to identify a protective antigen of HPG for development of a safe and effective vaccine against avian infectious coryza both as a component vaccine and as a vector vaccine.
Among protective antigens of HPG such as hemagglutinin (HA) and outer-membrane protein, HA is considered a most important antigen since immunization of chicken with HPG increased a hemagglutination-inhibition antibody (hereinafter referred to as “HI antibody”) and higher protective effect is observed for chickens with high level of HI antibody (Otsuki, et al. (1974); Kume, et al. (1984)).
Serotype of HPG is classified into serotypes A, B and C (Page, (1962)) or into serotypes 1 and 2 (Sawata, et al. (1978)) based on the agglutination test. It is considered that serotype A by Page corresponds to serotype 1 by Sawata, et al. whereas serotype C by Page corresponds to serotype 2 by Sawata, et al. (Kume, et al. (1980); Sawata, et al. (1980)).
Kume, et al. reported that HPG serotype A (serotype 1) has at least three kinds of HA, i.e. HA-L (heat-labile, trypsin-sensitive), HA-HL (heat-labile, trypsin-resistant) and HA-HS (heat-stable, trypsin-resistant), and that HA-L alone exhibits not only HA activity to usual fresh chicken erythrocytes but also to glutaraldehyde-fixed chicken erythrocytes and is involved in protection against infection with HPG serotype A (Kume, (1983); Sawata, et al. (1984)).
Iritani et al. reported that HPG serotype A has two kinds of HA, i.e. type 1 HA (heat-labile, protease-sensitive) and type 2 HA (heat-labile, protease-resistant), and that type 1 HA, which is heat-labile and protease-sensitive and consisted of a polypeptide having a molecular weight of about 39 kd as a subunit, is involved in protection against infection (Yamaguchi, et al. (1980); Iritani, et al. (1980)). It is considered that HA-L and HA-HL by Kume, et al. correspond to type 1 HA and type 2 HA by Iritani, et al., respectively. As to HPG serotype C (serotype 2), Sawata, et al. reported that an antigen was found which is heat-labile and trypsin-sensitive and exhibits the HA activity to glutaraldehyde-fixed chicken erythrocytes and that this antigen is distinct from HA of HPG serotype A in their antigenicity (Sawata, et al. (1982)). However, to date, a protective antigen of HPG has not yet been materially identified except for type 1 HA produced by HPG serotype A as reported by Iritani, et al.
As mentioned hereinabove, the conventional inactivated vaccine obtained by inactivating Haemophilus paragallinarum cells with thimerosal, formalin and the like has provoked problems in that the adverse side effects as mentioned above are induced when it is applied to fowls in a large amount since it includes various substances from the cells other than the protective antigen.